(1) Field of the Invention
The present invention relates to a heterogeneous or two step assay method and test kit for quantitatively determining thymosin alpha.sub.1. In particular the present invention relates to an enzyme imunossay (EIA) method for determining thymosin alpha.sub.1.
(2) Prior Art
Thymosin fraction 5 (TF5) is an extract of bovine thymus containing from 40 to 50 peptide components, 20 of which have been purified to homogeneity or near homogeneity (Goldstein et al., 1981). At least 5 of these peptides (thymosin alpha.sub.1, beta.sub.4, beta.sub.9, beta.sub.10, and beta.sub.11) have been sequenced, and several synthesized (Low et al., 1979, 1981, 1983; Wetzel et al., 1980). Thymosin alpha.sub.1, an acidic peptide with a MW of 3108, is a potent inducer of T cells and can influence immunoregulatory T cell function (Goldstein et al., 1981). TF5 and thymosin alpha.sub.1 promote the production of migration inhibitory factor in the mouse and guinea pig (Thruman et al., 1981; Neta and Salvin, 1983), the production of both alpha- and gamma-interferon in humans (Svedersky et al., 1982), and the enhancement of interleukin 2 production by human radioimmunoassay (RIA) has been developed to measure thymosin alpha.sub.1 in human serum. The antiseru to thymosin alpha.sub.1 was raised in rabbits against a chemically synthesized thymosin alpha.sub.1, prepared by solution synthesis (McClure et al., 1982; Naylor et al., 1984).
The prior art has described a number of assay methods for determining thymosin alpha.sub.1 including radio-, enzyme- and fluorescence-immunoassays. The RIA method involves the use of radioactive tracers incorporated into a modified thymosin-alpha.sub.1 which competes for an antibody along with the unknown amount of thymosin-alpha.sub.1 in solution. The amount of thymosin alpha.sub.1 is a function of the radioactivity in the thymosin alpha.sub.1 antibody complex which is isolated. The method is regarded as a homogeneous or one step method. This method is described by McClure et al in J. of Imnunology 128, 368-375 (1982), J. of Immunological Methods 60, 53-60 (1983) and in U.S. Pat. Nos. 4,264,571, 4,339,427 and 4,427,783 for instance U.S. Pat. No. 4,055,633 describes a related method. Such methods are accurate; however, the use of radioactive materials requires considerable chemical skills and also care to prevent exposure of the operator to the radioactivity. U.S. Pat. No. 4,315,907 describes a general immunoassay method.
The prior art has also described a homogeneous EIA method which uses an antibody which complexes with thymosin alpha.sub.1 in solution and with solid phase bound thymosin alpha.sub.1. The solid phase bound thymosin alpha.sub.1 competes with the liquid phase thymosin alpha.sub.1 for the antibody. The amount of thymosin alpha.sub.1 in the solution is related to the solid phase bound thymosin alpha.sub.1 and antibody complex formed which is measured. This method is described by Mutchnick et al in J. of Immunological Methods 60 53-60 (1983) and is referred to as an enzyme linked immunoassay (ELISA) method. It has been found that this method is not completely reliable due to variability in the complexing of the antibody to the thymosin alpha.sub.1 under the conditions described in this publication.